Human amine transporter

ABSTRACT

A Human amine transporter polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also provided are methods for detecting agonists and antagonists to such polypeptide and the use of agonists and antagonists for treating diseases related to the underexpression and over-expression of the Human amine transporter of the present invention. Also disclosed are methods for detecting mutations in the nucleic acid sequence encoding the polypeptide and for detecting altered levels of the soluble form of the polypeptide.

[0001] This application is a divisional of U.S. application Ser. No.09/502,018, filed Feb. 11, 2000; which is a divisional of U.S.application Ser. No. 09/139,675, filed Aug. 25, 1998, now U.S. Pat. No.6,117,426; which is a divisional of U.S. application Ser. No.08/471,496, filed Jun. 6, 1995, now U.S. Pat. No. 5,798,223; which is aContinuation-in-Part of PCT/US95/02645, filed Mar. 1, 1995. The fulldisclosures of each of these is herein incorporated by reference.

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention is a human amine transporter. The invention alsorelates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

[0003] Neurosensory and neuromotor functions are carried out byneurotransmission. Neurotransmission is the conductance of a nerveimpulse from one neuron, called the presynaptic neuron, to anotherneuron, called the postsynaptic neuron, across the synaptic cleft.Transmission of the nerve impulse across the synaptic cleft involves thesecretion of neurotransmitter substances. The neurotransmitter ispackaged into vesicles in the presynaptic neuron and released into thesynaptic cleft to find its receptor at the postsynaptic neuron.Transmission of the nerve impulse is normally transient.

[0004] An essential property of synaptic transmission is the rapidtermination of action following neurotransmitter release. For manyneurotransmitters, including catecholamine, serotonin, and certain aminoacids (e.g., gamma-aminobutyric acid (GABA), glutamate and glycine),rapid termination of synaptic action is achieved by the uptake of theneurotransmitter into the presynaptic terminal and surrounding glialcells. This rapid re-accumulation of a neurotransmitter is the result ofre-uptake by the presynaptic terminals.

[0005] At presynaptic terminals, the various molecular structures forre-uptake are highly specific for such neurotransmitters as choline andthe biogenic amines (low molecular weight neurotransmitter substancessuch as dopamine, norepinephrine, epinephrine, serotonin and histamine).These molecular apparatuses are termed transporters. These transportersmove neurotransmitter substances from the synaptic cleft back across thecell membrane of the presynaptic neuron into the cytoplasm of thepresynaptic terminus and therefore terminate the function of thesesubstances. Inhibition or stimulation of neurotransmitter uptakeprovides a means for modulating the effects of the endogenousneurotransmitters.

[0006] Re-uptake of neurotransmitter substances by the transporters maybe sodium-dependent. For instance, the GABA transporter is a member ofthe recently described sodium-dependent neurotransmitter transportergene family. These transporters are transmembrane receptor complexeshaving an extracellular portion, a transmembrane portion and anintracellular portion. A significant degree of homology exists in thetransmembrane domains of the entire family of sodium-dependentneurotransmitter transporter proteins, with considerable stretches ofidentical amino acids, while much less homology is apparent in theintracellular and extracellular loops connecting these domains. Theextracellular loop in particular seems to be unique for eachtransporter. This region may contribute to substrate and/or inhibitorspecificities.

[0007] Identifying the novel amine transporter of the present inventionand elucidating the structural and functional distinctions betweendifferent types of transporters is important in understanding thecellular and molecular bases of behavior and disease.

SUMMARY OF THE INVENTION

[0008] The polypeptide of the present invention has been putativelyidentified as an amine transporter. This identification has been made asa result of amino acid sequence homology to the rat amine transporter.

[0009] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is a human amine transporter,as well as biologically active and diagnostically or therapeuticallyuseful fragments, analogs and derivatives thereof.

[0010] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding a human aminetransporter, including mRNAs, DNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments and derivatives thereof.

[0011] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human amine transporternucleic acid sequence, under conditions promoting expression of saidprotein and subsequent recovery of said protein.

[0012] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide for screening for agonists andantagonists and ligands to such polypeptide.

[0013] In accordance with yet a further aspect of the present invention,there is provided a method for utilizing such agonists for stimulatingthe amine transporter uptake of neurotransmitter ligands for thetreatment of diseases related to under-expression of the aminetransporter or over-expression of the ligand.

[0014] In accordance with yet another aspect of the present invention,there is also provided a process for using antagonists for inhibitingthe amine transporter uptake of neurotransmitter ligands for thetreatment of diseases related to over-expression of the aminetransporter or under-expression of the ligand.

[0015] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0016] In accordance with yet a further aspect of the present invention,there are also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to human aminetransporter sequences.

[0017] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe under-expression and over-expression of the amine transporterpolypeptide and mutations in the nucleic acid sequences encoding suchpolypeptide.

[0018] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0019] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

[0020] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0021] FIGS. 1A-1E illustrate the cDNA sequence (SEQ ID NO:1) andcorresponding deduced amino acid sequence (SEQ ID NO:2) of the humanamine transporter of the present invention. The standard one-letterabbreviations for amino acids are used. Sequencing was performed using a373 Automated DNA sequencer (Applied Biosystems, Inc.). Sequencingaccuracy is predicted to be greater than 97% accurate.

[0022] FIGS. 2A-2B illustrate an amino acid homology alignment betweenthe amine transporter (SEQ ID NO:2) and a rat amine transporter (SEQ IDNO:9) (retrieved from Genbank public database).

DETAILED DESCRIPTION OF THE INVENTION

[0023] The amine transporter of the present invention may be responsiblefor re-uptake of one or any of the amine neurotransmitters present inmammalian cells. Examples of such amine transporters include dopamine,norepinephrine, epinephrine, serotonin and histamine, and other aminoacid transmitters, including GABA, glycine and glutamate.

[0024] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIGS. 1A-1E(SEQ ID No. 2) or for the mature polypeptide encoded by the cDNA of theclone deposited as ATCC Deposit No. 75980 on Dec. 16, 1994 at theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209.

[0025] A polynucleotide encoding a polypeptide of the present inventionmay be obtained from a variety of human tissues. The polynucleotide ofthis invention was discovered in a cDNA library derived from a humanadrenal gland tumor. It is structurally related to the amine transporterfamily. It contains an open reading frame encoding a protein of 470amino acid residues. The protein exhibits the highest degree of homologyto the rat amine transporter with 80% identity and 86% similarity over a468 amino acid stretch.

[0026] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIGS. 1A-1E (SEQ ID No. 2) orthat of the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIGS.1A-1E (SEQ ID No. 2) or the deposited cDNA.

[0027] The polynucleotide which encodes for the mature polypeptide ofFIGS. 1A-1E (SEQ ID No. 2) or for the mature polypeptide encoded by thedeposited cDNA may include only the coding sequence for the maturepolypeptide or the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0028] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0029] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIGS. 1A-1E (SEQ ID No. 2) or the polypeptide encoded by thecDNA of the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0030] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIGS. 1A-1E (SEQ ID No. 2) or thesame mature polypeptide encoded by the cDNA of the deposited clone aswell as variants of such polynucleotides which variants encode for afragment, derivative or analog of the polypeptide of FIGS. 1A-1E (SEQ IDNo. 2) or the polypeptide encoded by the cDNA of the deposited clone.Such nucleotide variants include deletion variants, substitutionvariants and addition or insertion variants.

[0031] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIGS. 1A-1E (SEQ ID No. 1) or of the coding sequenceof the deposited clone. As known in the art, an allelic variant is analternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

[0032] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0033] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0034] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0035] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least85%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIGS. 1A-1E (SEQ ID NO:1)or the deposited cDNA(s).

[0036] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0037] Thus, the present invention is directed to polynucleotides havingat least a 85% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0038] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0039] The present invention further relates to a human aminetransporter polypeptide which has the deduced amino acid sequence ofFIGS. 1A-1E (SEQ ID No. 2) or which has the amino acid sequence encodedby the deposited cDNA, as well as fragments, analogs and derivatives ofsuch polypeptide.

[0040] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIGS. 1A-1E (SEQ ID No. 2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0041] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0042] The fragment, derivative or analog of the polypeptide of FIGS.1A-E (SEQ ID No. 2) or that encoded by the deposited cDNA may be (i) onein which one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol). Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

[0043] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0044] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0045] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 80% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0046] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0047] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0048] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0049] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the human amine transporter genes. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0050] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0051] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0052] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0053] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0054] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0055] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera SJ9; animal cells such as CHO, HEK, COS orBowes melanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0056] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0057] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lacd, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0058] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0059] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0060] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0061] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0062] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

[0063] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0064] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0065] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0066] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0067] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0068] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HEK, HeLa and BHKcell lines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0069] The human amine transporter polypeptide can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

[0070] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0071] Fragments of the full length human amine transporter gene may beused as a hybridization probe for a cDNA library to isolate the fulllength gene and to isolate other genes which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype generally have at least 20 bases. Preferably, however, the probeshave at least 30 bases and generally do not exceed 50 bases, althoughthey may have a greater number of bases. The probe may also be used toidentify a cDNA clone corresponding to a full length transcript and agenomic clone or clones that contain the complete human aminetransporter gene including regulatory and promotor regions, exons, andintrons. As an example of a screen comprises isolating the coding regionof the human amine transporter gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0072] This invention provides a method for determining amineneurotransmitters which are transported by the human amine transporterof the present invention. An example of an assay which will identifythese neurotransmitters comprises infecting mammalian cells withrecombinant vaccinia virus strain VTF-7 encoding a T7 RNA polymerase andfollowing such infection with liposome-mediated transfection with theamine transporter gene through the use of a vector, for example,pBSSKII(−). Controlled transfections are also done with equivalentamounts of vector alone. Assays are performed eight hours followingtransfection in modified Krebs-Ringer-HEPES buffer. Cells are thenincubated with [³H] neurotransmitter (for example, GABA, dopamine,serotonin, etc.). Uptake is stopped by placing the cells on ice. Cellsare solubilized in one percent SDS, and the amount of radioactivityaccumulated is determined by liquid scintillation counting. Asignificant amount of uptake determines that the particularneurotransmitter is taken up by the human amine transporter of thepresent invention by determining background using control transfectionswith pBSSKII for each assay and subtracting the values obtained from thesignals determined for the specific amine neurotransmitters.

[0073] This invention also provides a method of detecting expression ofan amine transporter on the surface of a cell by detecting the presenceof mRNA coding for an amine transporter. This method comprises obtainingtotal mRNA from the cell using methods well-known in the art andcontacting the mRNA so obtained with a nucleic acid probe of at least 15nucleotides and which is capable of specifically hybridizing with asequence included within the sequence of a nucleic acid moleculeencoding a human amine transporter, under hybridizing conditions,detecting the presence of mRNA hybridized to the probe, and therebydetecting the expression of the amine transporter by the cell.Hybridization of probes to target nucleic acid molecules such as mRNAmolecules employs techniques well known in the art. However, in oneembodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing a column which binds the poly-A tails of the mRNA molecules. ThemRNA is then exposed to radioactively labelled probe on a nitrocellulosemembrane, and the probe hybridizes to and thereby labels complementarymRNA sequences. Binding may be detected by autoradiography orscintillation counting. However, other methods for performing thesesteps are well known to those of skill in the art.

[0074] Alternatively, an antibody directed to the human aminetransporter may be employed under conditions permitting binding of theantibody to the transporter, and detecting the presence of thetransporter on the surface of the cell. Such a method may be employedfor determining whether a given cell is defective in expression of theamine transporter. Detection methods include fluorescent markers boundto the antibodies.

[0075] The invention also provides a method for determining whether acompound not known to be capable of specifically binding to a humanamine transporter can specifically bind to the human amine transporter,which comprises contacting a mammalian cell comprising a plasmid adaptedfor expression in a mammalian cell which plasmid further comprises a DNAwhich expresses the amine transporter on the cell surface with thecompound under conditions permitting binding of ligands known to bind tothe amine transporter, detecting the presence of any compound bound tothe mammalian amine transporter, the presence of bound compoundindicating that the compound is capable of specifically binding to thehuman amine transporter.

[0076] This invention also provides a method of screening drugs toidentify drugs which specifically interact with, and bind to a humanamine transporter on the surface of a cell which comprises contacting amammalian cell which expresses the human amine transporter on thesurface of a cell with a plurality of drugs, detecting those drugs whichbind to the cell, and thereby identifying drugs which specificallyinteract with, and bind to, the human amine transporter.

[0077] The present invention further provides a method for identifyingagonist or antagonist compounds to the human amine transporter of thepresent invention by the employment of competition assays. An example ofsuch an assay for identifying antagonists comprises contacting aneuronal cell which expresses the human amine transporter on the surfacethereof with a known neurotransmitter, in the presence of a potentialcompound to determine the amount of neurotransmitter transported.Controls may also be prepared in the absence of the potential compoundand the amount of amine neurotransmitter transported by the cell uponcomparison to the control cell indicates if the potential compoundstimulated transport or inhibited transport of the labeled amineneurotransmitter by the transfected mammalian cell.

[0078] Examples of human amine transporter antagonists include anantibody directed to the human amine transporter which comprises, forexample, a monoclonal antibody directed to an epitope of a human aminetransporter present on the surface of the cell. These antibodies areuseful to detect the presence of human amine transporters or to inhibitthe function of the transporters in humans.

[0079] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix--see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of human aminetransporter. The antisense RNA oligonucleotide hybridizes to the mRNA invivo and blocks translation of the mRNA molecule into the human aminetransporter polypeptide (antisense-Okano, J. Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). The oligonucleotides described abovecan also be delivered to cells such that the antisense RNA or DNA may beexpressed in vivo to inhibit production of human amine transporter.

[0080] Potential antagonists also include a soluble form of a humanamine transporter, e.g. a fragment of the transporter, which binds tothe neurotransmitter and prevents it from interacting with the humanamine transporter.

[0081] Potential antagonists further include a small molecule whichbinds to and occupies the extracellular portion of the human aminetransporter thereby making the human amine transporter inaccessible tothe neurotransmitter such that transport is inhibited. Examples of smallmolecules include but are not limited to small peptides or peptide-likemolecules.

[0082] This invention additionally provides a method of treating anabnormal condition related to an excess of amine transporter activitywhich comprises administering to a subject the antagonist as hereinabovedescribed along with a pharmaceutically acceptable carrier in an amounteffective to block binding of naturally occurring substrates to theamine transporters and thereby alleviate the abnormal condition.Examples of abnormal conditions include epilepsy, schizophrenia,depression, cognitive impairment, anxiety and migraine headaches.

[0083] The invention also provides a method of treating abnormalconditions related to an under-expression of amine transporter activitywhich comprises administering to a subject an amount of the agonistdescribed above in combination with a pharmaceutically acceptablecarrier, in an amount effective to enhance binding of naturallyoccurring substrates to the amine transporter and thereby alleviate theabnormal conditions. Some examples of abnormal conditions areParkinson's disease and Alzheimer's disease.

[0084] The soluble form of the human amine transporter, and agonists andantagonists may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the transporter, agonist or antagonist and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0085] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such containers can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0086] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0087] The human amine transporter and agonists and antagonists whichare polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0088] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0089] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

[0090] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0091] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and beta-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0092] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the beta-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter whichcontrols the gene encoding the polypeptide.

[0093] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, psi-2, psi-AM, PA12, T19-14X, VT-19-17-H2, psiCRE, psiCRIP,GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, HumanGene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0094] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0095] This invention is also related to the use of the human aminetransporter gene as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thehuman amine transporter genes. Such diseases are related tounder-expression of the human amine transporter.

[0096] Individuals carrying mutations in the human amine transportergene may be detected at the DNA level by a variety of techniques.Nucleic acids for diagnosis may be obtained from a patient's cells, suchas from blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding thehuman amine transporter protein can be used to identify and analyzehuman amine transporter mutations. For example, deletions and insertionscan be detected by a change in size of the amplified product incomparison to the normal genotype. Point mutations can be identified byhybridizing amplified DNA to radiolabeled human amine transporter RNA oralternatively, radiolabeled human amine transporter antisense DNAsequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0097] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments' are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0098] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0099] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0100] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0101] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0102] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0103] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0104] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

[0105] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0106] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0107] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0108] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0109] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0110] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0111] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0112] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0113] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0114] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0115] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 degree C. are ordinarily used, but may vary in accordancewith the supplier's instructions. After digestion the reaction iselectrophoresed directly on a polyacrylamide gel to isolate the desiredfragment.

[0116] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0117] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in, the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0118] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0119] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLES Example 1 Bacterial Expression and Purification of Human AmineTransporter

[0120] The DNA sequence encoding human amine transporter, ATCC #75980,is initially amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ end sequences of the processed amine transporternucleic acid sequence (minus the signal peptide sequence). Additionalnucleotides corresponding to amine transporter gene are added to the 5′and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GACTAAAGCTTAATGCTCCGGCCCATTCTG 3′ (SEQ ID No. 3) contains aHindIII restriction enzyme site followed by 18 nucleotides of humanamine transporter coding sequence starting from the presumed terminalamino acid of the processed protein. The 3′ sequence 5′GAACTTCTAGACGGTCAGCCATGGTGACTGG 3′ (SEQ ID No. 4) contains complementarysequences to an XbaI site and is followed by 20 nucleotides of the humanamine transporter gene. The restriction enzyme sites correspond to therestriction enzyme sites on the bacterial expression vector pQE-9(Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibiotic resistance(Ampr), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 is then digested with HindIII and XbaI.The amplified sequences are ligated into pQE-9 and are inserted in framewith the sequence encoding for the histidine tag and the RBS. Theligation mixture is then used to transform E. coli strain M15/rep 4(Qiagen, Inc.) by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). M15/rep4 contains multiple copies of the plasmid pREP4, whichexpresses the lacd repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacd repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedhuman amine transporter is purified from this solution by chromatographyon a Nickel-Chelate column under conditions that allow for tight bindingby proteins containing the 6-His tag (Hochuli, E. et al., J.Chromatography 411:177-184 (1984)). Human amine transporter protein iseluted from the column in 6 molar guanidine HCl pH 5.0. and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinis dialyzed to 10 mmolar sodium phosphate.

Example 2 Cloning and Expression of Human Amine Transporter Using theBaculovirus Expression System

[0121] The DNA sequence encoding the full length human amine transporterprotein, ATCC #75980, is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

[0122] The 5′ primer has the sequence 5° CGGGATCCCTCCATGGCTCCGGCCCATTCTG 3′ (SEQ ID No. 5) and contains a BamHI restriction enzymesite (in bold) followed by 4 nucleotides resembling an efficient signalfor the initiation of translation in eukaryotic cells (Kozak, M., J.Mol. Biol., 196:947-950 (1987) which is just behind the first 18nucleotides of the human amine transporter gene (the initiation codonfor translation “ATG” is underlined).

[0123] The 3′ primer has the sequence 5′ CGGGATCCCGCT CAGCCATGGTGACTGGT3′ (SEQ ID No. 6) and contains the cleavage site for the restrictionendonuclease BamHI and 18 nucleotides complementary to the 3′non-translated sequence of the human amine transporter gene. Theamplified sequences are isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment is then digested with the endonucleases BamHI andthen purified again on a it agarose gel. This fragment is designated F2.

[0124] The vector pRG1 (modification of pVL941 vector, discussed below)is used for the expression of the human amine transporter protein usingthe baculovirus expression system (for review see: Summers, M. D. andSmith, G. E. 1987, A manual of methods for baculovirus vectors andinsect cell culture procedures, Texas Agricultural Experimental StationBulletin No. 1555). This expression vector contains the strongpolyhedrin promoter of the Autographa califomica nuclear polyhedrosisvirus (AcMNPV) followed by the recognition sites for the restrictionendonucleases BamHI. The polyadenylation site of the simian virus (SV)40is used for efficient polyadenylation. For an easy selection ofrecombinant viruses the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0125] The plasmid is digested with the restriction enzymes BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA is then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

[0126] Fragment F2 and the dephosphorylated plasmid V2 are ligated withT4 DNA ligase. E. coli HB101 cells are then transformed and bacteriaidentified that contained the plasmid (pBac-Human amine transporter)with the human amine transporter gene using the enzyme BamHI. Thesequence of the cloned fragment is confirmed by DNA sequencing.

[0127] 5 μg of the plasmid pBac-Human amine transporter isco-transfected with 1.0 μg of a commercially available linearizedbaculovirus (“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego,Calif.) using the lipofection method (Felgner et al. Proc. Natl. Acad.Sci. USA, 84:7413-7417 (1987)).

[0128] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac-Humanamine transporter are mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added dropwise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27 degree C. After 5 hours the transfection solution is removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. The plate is put back into an incubator and cultivationcontinued at 27 degree C. for four days.

[0129] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0130] Four days after the serial dilution, the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4 degree C.

[0131] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-Human amine transporter at a multiplicity of infection(MOI) of 2. Six hours later the medium is removed and replaced withSF900 II medium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) are added. The cells are further incubated for 16hours before they are harvested by centrifugation and the labelledproteins visualized by SDS-PAGE and autoradiography.

Example 3 Expression of Recombinant Human Amine Transporter in COS Cells

[0132] The expression of plasmid, Human amine transporter HA is derivedfrom a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin ofreplication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, a SV40 intronand polyadenylation site. A DNA fragment encoding the entire Human aminetransporter precursor and a HA tag fused in frame to its 3′ end iscloned into the polylinker region of the vector, therefore, therecombinant protein expression is directed under the CMV promoter. TheHA tag correspond to an epitope derived from the influenza hemagglutininprotein as previously described (I. Wilson, et al., Cell, 37:767,(1984)). The infusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

[0133] The plasmid construction strategy is described as follows: TheDNA sequence encoding Human amine transporter, ATCC #75980, isconstructed by PCR using two primers: the 5′ primer 5′GTCCAAGCTTGCCACCATGCTGCGGCCCATTCTG 3′ (SEQ ID No. 7) contains a HindIIIsite followed by 18 nucleotides of Human amine transporter codingsequence starting from the initiation codon; the 3′ sequence 5′CTAGCTCGAGTCAGCCATGGTGACTGGTAGCGTAGTCTGGGACGTCGTATGGGT AGCA 3′ (SEQ IDNo. 8) contains complementary sequences to an XhoI site, translationstop codon, HA tag and the last 18 nucleotides of the Human aminetransporter coding sequence (not including the stop codon). Therefore,the PCR product contains a HindIII site, human amine transporter codingsequence followed by HA tag fused in frame, a translation terminationstop codon next to the HA tag, and an HindIII site. The PCR amplifiedDNA fragment and the vector, pcDNAI/Amp, are digested with HindIII andXhoI restriction enzyme and ligated. The ligation mixture is transformedinto E. coli strain SURE (Stratagene Cloning Systems, La Jolla, Calif.)the transformed culture is plated on ampicillin media plates andresistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant aminetransporter, COS cells are transfected with the expression vector byDRAB-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the Human amine transporter HA protein is detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media is then collected and cells are lysed withdetergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5%DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Bothcell lysate and culture media are precipitated with a HA specificmonoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGEgels.

Example 4 Expression Pattern of Human Amine Transporter in Human Tissue

[0134] Northern blot analysis is carried out to examine the levels ofexpression of Human amine transporter in human tissues. Total cellularRNA samples are isolated with RNAzol™ B system (Biotecx Laboratories,Inc. Houston, Tex.). About 10 =82 g of total RNA isolated from eachhuman tissue specified is separated on 1% agarose gel and blotted onto anylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning, ColdSpring Harbor Press, (1989)). The labeling reaction is done according tothe Stratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G-50 column (5 Prime-3 Prime, Inc. Boulder,Colo.). The filter is then hybridized with radioactive labeled fulllength Human amine transporter gene at 1,000,000 cpm/ml in 0.5 M NaPO₄,pH 7.4 and 7% SDS overnight at 65 degree C. After wash twice at roomtemperature and twice at 60 degree C. with 0.5×SSC, 0.1% SDS, the filteris then exposed at −70 degree C. overnight with an intensifying screen.

Example 5 Expression Via Gene Therapy

[0135] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37 degree C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0136] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0137] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer $further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified $EcoRIand HindIII fragment are added together, in the presence of T4 DNAligase. The resulting mixture is maintained under conditions appropriatefor ligation of the two fragments. The ligation mixture is used totransform bacteria HB101, which are then plated onto agar-containingkanamycin for the purpose of confirming that the vector had the gene ofinterest properly inserted.

[0138] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0139] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0140] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0141] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 9 1 2885 DNA Homo sapiens CDS (719)..(2128) UNSURE (39) May be anynucleic acid 1 tcctgcgtta tccccctgat tctgtggata accgtattnc cgcctttgagtgagctgata 60 ccgctcnccn cagccgaacg accgagcgca gcgagtcagt gagcgaggaagcggaagagc 120 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgcagctggcacg 180 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtgagttagctca 240 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttgtgtggaattg 300 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgccaagctcgaaa 360 ttaaccctca ctaaagggaa caaaagctgg agctccaccg cggtggcgnccgctctagaa 420 ctagtggatc ccccggnctg caggggcaca cacacgcaca catacacagaatcctcagat 480 aacaggaggc aataaatcca acagcacatc cacgttcaga gaacagtgtccctgctgtct 540 tgctaacagc tgccaatacc tcactgagtg cctcacacca acatgggctccaagtgagtt 600 tcattcgtct gggcagactc cctcccctct tccataaagg ctgcaggagacctgtagctg 660 tcacaggacc ttccctaaga gcccgcaggg ggaagactgc cccagtccggccatcacc 718 atg ctc cgg ccc att ctg gat gct ccc cag cgg ttg ctg aag gagggg 766 Met Leu Arg Pro Ile Leu Asp Ala Pro Gln Arg Leu Leu Lys Glu Gly1 5 10 15 aga gcg tcc cgg cag ctg gtg ctg gtg gtg gta ttc gtc gct ttgctc 814 Arg Ala Ser Arg Gln Leu Val Leu Val Val Val Phe Val Ala Leu Leu20 25 30 ctg gac aac atg ctg ttt act gtg gtg gtg cca att gtg ccc acc ttc862 Leu Asp Asn Met Leu Phe Thr Val Val Val Pro Ile Val Pro Thr Phe 3540 45 cta tat gac atg gag ttc aaa gaa gtc atc tct tct ctg cac ctc ggg910 Leu Tyr Asp Met Glu Phe Lys Glu Val Ile Ser Ser Leu His Leu Gly 5055 60 cat gcc gga agt tcc cca cat gcc ctc gcc tct cct gcc ttt tcc acc958 His Ala Gly Ser Ser Pro His Ala Leu Ala Ser Pro Ala Phe Ser Thr 6570 75 80 atc ttc tcc ttc ttc aac aac aac acc gtg gct gtt gaa gaa agc gta1006 Ile Phe Ser Phe Phe Asn Asn Asn Thr Val Ala Val Glu Glu Ser Val 8590 95 cct agt gga ata gca tgg atg aat gac act gcc agc acc atc cca cct1054 Pro Ser Gly Ile Ala Trp Met Asn Asp Thr Ala Ser Thr Ile Pro Pro 100105 110 cca gcc act gaa gcc atc tca gct cat aaa aac aac tgc ttg caa ggc1102 Pro Ala Thr Glu Ala Ile Ser Ala His Lys Asn Asn Cys Leu Gln Gly 115120 125 aca ggt ttc ttg gag gaa gag act acc cgg gtc ggg gtt ctg ttt gct1150 Thr Gly Phe Leu Glu Glu Glu Thr Thr Arg Val Gly Val Leu Phe Ala 130135 140 tca aag gct gtg atg caa ctt ctg gtc aac cca ttc gtg ggc cct ctc1198 Ser Lys Ala Val Met Gln Leu Leu Val Asn Pro Phe Val Gly Pro Leu 145150 155 160 acc aac agg att gga tat cat atc ccc atg ttt gct ggc ttt gttatc 1246 Thr Asn Arg Ile Gly Tyr His Ile Pro Met Phe Ala Gly Phe Val Ile165 170 175 atg ttt ctc tcc aca gtt atg ttt gct ttt tct ggg acc tat actcta 1294 Met Phe Leu Ser Thr Val Met Phe Ala Phe Ser Gly Thr Tyr Thr Leu180 185 190 ctc ttt gtg gcc cga acc ctt caa ggc att gga tct tca ttt tcatct 1342 Leu Phe Val Ala Arg Thr Leu Gln Gly Ile Gly Ser Ser Phe Ser Ser195 200 205 gtt gca ggt ctt gga atg ctg gcc agt gtc tac act gat gac catgag 1390 Val Ala Gly Leu Gly Met Leu Ala Ser Val Tyr Thr Asp Asp His Glu210 215 220 aga gga cga gcc atg gga act gct ctg ggg ggc ctg gcc ttg gggttg 1438 Arg Gly Arg Ala Met Gly Thr Ala Leu Gly Gly Leu Ala Leu Gly Leu225 230 235 240 ctg gtg gga gct ccc ttt gga agt gta atg tac gag ttt gttggg aag 1486 Leu Val Gly Ala Pro Phe Gly Ser Val Met Tyr Glu Phe Val GlyLys 245 250 255 tct gca ccc ttc ctc atc ctg gcc ttc ctg gca cta ctg gatgga gca 1534 Ser Ala Pro Phe Leu Ile Leu Ala Phe Leu Ala Leu Leu Asp GlyAla 260 265 270 ctc cag ctt tgc atc cta cag cct tcc aaa gtc tct cct gagagt gcc 1582 Leu Gln Leu Cys Ile Leu Gln Pro Ser Lys Val Ser Pro Glu SerAla 275 280 285 aag ggg act ccc ctc ttt atg ctt ctc aaa gac cct tac atcctg gtg 1630 Lys Gly Thr Pro Leu Phe Met Leu Leu Lys Asp Pro Tyr Ile LeuVal 290 295 300 gct gca ggg tcc atc tgc ttt gcc aac atg ggg gtg gcc atcctg gag 1678 Ala Ala Gly Ser Ile Cys Phe Ala Asn Met Gly Val Ala Ile LeuGlu 305 310 315 320 ccc aca ctg ccc atc tgg atg atg cag acc atg tgc tccccc aag tgg 1726 Pro Thr Leu Pro Ile Trp Met Met Gln Thr Met Cys Ser ProLys Trp 325 330 335 cag ctg ggt cta gct ttc ttg cct gcc agt gtg tcc tacctc att ggc 1774 Gln Leu Gly Leu Ala Phe Leu Pro Ala Ser Val Ser Tyr LeuIle Gly 340 345 350 acc aac ctc ttt ggt gtg ttg gcc aac aag atg ggt cggtgg ctg tgt 1822 Thr Asn Leu Phe Gly Val Leu Ala Asn Lys Met Gly Arg TrpLeu Cys 355 360 365 tcc cta atc ggg atg ctg gta gta ggt acc agc ttg ctctgt gtt cct 1870 Ser Leu Ile Gly Met Leu Val Val Gly Thr Ser Leu Leu CysVal Pro 370 375 380 ctg gct cac aaa aat ttt ggt ctc att ggc ccc aat gcaggg ctt ggc 1918 Leu Ala His Lys Asn Phe Gly Leu Ile Gly Pro Asn Ala GlyLeu Gly 385 390 395 400 ctt ncc ata ggc atg gtg gaa tct tct atg atg cccatc atg ggg cac 1966 Leu Xaa Ile Gly Met Val Glu Ser Ser Met Met Pro IleMet Gly His 405 410 415 ctg gtg gat cca cgc cac acc tcg gtg tat ggg agtgtc cac gcc atc 2014 Leu Val Asp Pro Arg His Thr Ser Val Tyr Gly Ser ValHis Ala Ile 420 425 430 gct gat gtg gct ttt tgc atg ggc ttt gct ata ggctat tct gag tca 2062 Ala Asp Val Ala Phe Cys Met Gly Phe Ala Ile Gly TyrSer Glu Ser 435 440 445 gga ctg ccc cat gga gac ccg gat gta tca acc cagaaa cct ctt ccc 2110 Gly Leu Pro His Gly Asp Pro Asp Val Ser Thr Gln LysPro Leu Pro 450 455 460 tgg acc agt cac cat ggc tgacccacgg ctcagtggcctcaaaacctc 2158 Trp Thr Ser His His Gly 465 470 tgcctgggat cttcttcctcccctcccatg gagactgtcc ctcatactct tctcacctgt 2218 gtaacttgta gctcttcmtctatgccttgg tgccgcagtg gcccatcttt tatgggaaga 2278 cagagtgatg caccyycccgctgctgtgag gttgattaaa cttgagctgt gacggggttc 2338 tgcaaggggt gactcattgyatagaggtgg tagtgagtaa tgtgcccctg aaaccagtgg 2398 ggtgactgac aagcctctttaatctgttgc ctgattttct ctggcatagc cccaacagat 2458 cggaagagtg ttaccctctttwccctcaac gtgttctttc ccgggttttc cccagccgag 2518 ttgagaaaat gttctcagcattgtcttgct gccaaatgcc agcktgaaga gttwggtatg 2578 ktttttctnc catttattttatttattwac taaagtgaat gattttactg tggytaaatc 2638 tagagctgct aaaagggctttaccctcagt gaaaagtgtc ttctatttnc atwatctttc 2698 agaaacwgga gcccatttctcttctggtgg agttatngac atcctcctga ccncccctgt 2758 gtntncctac ctntactgaacctcttagac tctnagaaat aaaagtagaa gaaagacaga 2818 aaaattaact gattagacccaagatttcat gggaagaagt taaaagaaac tgccttggaa 2878 atccctc 2885 2 470 PRTHomo sapiens UNSURE (402) May be any amino acid 2 Met Leu Arg Pro IleLeu Asp Ala Pro Gln Arg Leu Leu Lys Glu Gly 1 5 10 15 Arg Ala Ser ArgGln Leu Val Leu Val Val Val Phe Val Ala Leu Leu 20 25 30 Leu Asp Asn MetLeu Phe Thr Val Val Val Pro Ile Val Pro Thr Phe 35 40 45 Leu Tyr Asp MetGlu Phe Lys Glu Val Ile Ser Ser Leu His Leu Gly 50 55 60 His Ala Gly SerSer Pro His Ala Leu Ala Ser Pro Ala Phe Ser Thr 65 70 75 80 Ile Phe SerPhe Phe Asn Asn Asn Thr Val Ala Val Glu Glu Ser Val 85 90 95 Pro Ser GlyIle Ala Trp Met Asn Asp Thr Ala Ser Thr Ile Pro Pro 100 105 110 Pro AlaThr Glu Ala Ile Ser Ala His Lys Asn Asn Cys Leu Gln Gly 115 120 125 ThrGly Phe Leu Glu Glu Glu Thr Thr Arg Val Gly Val Leu Phe Ala 130 135 140Ser Lys Ala Val Met Gln Leu Leu Val Asn Pro Phe Val Gly Pro Leu 145 150155 160 Thr Asn Arg Ile Gly Tyr His Ile Pro Met Phe Ala Gly Phe Val Ile165 170 175 Met Phe Leu Ser Thr Val Met Phe Ala Phe Ser Gly Thr Tyr ThrLeu 180 185 190 Leu Phe Val Ala Arg Thr Leu Gln Gly Ile Gly Ser Ser PheSer Ser 195 200 205 Val Ala Gly Leu Gly Met Leu Ala Ser Val Tyr Thr AspAsp His Glu 210 215 220 Arg Gly Arg Ala Met Gly Thr Ala Leu Gly Gly LeuAla Leu Gly Leu 225 230 235 240 Leu Val Gly Ala Pro Phe Gly Ser Val MetTyr Glu Phe Val Gly Lys 245 250 255 Ser Ala Pro Phe Leu Ile Leu Ala PheLeu Ala Leu Leu Asp Gly Ala 260 265 270 Leu Gln Leu Cys Ile Leu Gln ProSer Lys Val Ser Pro Glu Ser Ala 275 280 285 Lys Gly Thr Pro Leu Phe MetLeu Leu Lys Asp Pro Tyr Ile Leu Val 290 295 300 Ala Ala Gly Ser Ile CysPhe Ala Asn Met Gly Val Ala Ile Leu Glu 305 310 315 320 Pro Thr Leu ProIle Trp Met Met Gln Thr Met Cys Ser Pro Lys Trp 325 330 335 Gln Leu GlyLeu Ala Phe Leu Pro Ala Ser Val Ser Tyr Leu Ile Gly 340 345 350 Thr AsnLeu Phe Gly Val Leu Ala Asn Lys Met Gly Arg Trp Leu Cys 355 360 365 SerLeu Ile Gly Met Leu Val Val Gly Thr Ser Leu Leu Cys Val Pro 370 375 380Leu Ala His Lys Asn Phe Gly Leu Ile Gly Pro Asn Ala Gly Leu Gly 385 390395 400 Leu Xaa Ile Gly Met Val Glu Ser Ser Met Met Pro Ile Met Gly His405 410 415 Leu Val Asp Pro Arg His Thr Ser Val Tyr Gly Ser Val His AlaIle 420 425 430 Ala Asp Val Ala Phe Cys Met Gly Phe Ala Ile Gly Tyr SerGlu Ser 435 440 445 Gly Leu Pro His Gly Asp Pro Asp Val Ser Thr Gln LysPro Leu Pro 450 455 460 Trp Thr Ser His His Gly 465 470 3 30 DNA Homosapiens 3 gactaaagct taatgctccg gcccattctg 30 4 31 DNA Homo sapiens 4gaacttctag acggtcagcc atggtgactg g 31 5 31 DNA Homo sapiens 5 cgggatccctccatggctcc ggcccattct g 31 6 29 DNA Homo sapiens 6 cgggatcccg ctcagccatggtgactggt 29 7 34 DNA Homo sapiens 7 gtccaagctt gccaccatgc tgcggcccattctg 34 8 58 DNA Homo sapiens 8 ctagctcgag tcagccatgg tgactggtagcgtagtctgg gacgtcgtat gggtagca 58 9 465 PRT Rattus sp. 9 Met Leu Gln ValVal Leu Gly Ala Pro Gln Arg Leu Leu Lys Glu Gly 1 5 10 15 Arg Gln SerArg Lys Leu Val Leu Val Val Val Phe Val Ala Leu Leu 20 25 30 Leu Asp AsnMet Leu Leu Thr Val Val Val Pro Ile Val Pro Thr Phe 35 40 45 Leu Tyr AlaThr Glu Phe Lys Asp Ser Asn Ser Ser Leu His Arg Gly 50 55 60 Pro Ser ValSer Ser Gln Gln Ala Leu Thr Ser Pro Ala Phe Ser Thr 65 70 75 80 Ile PheSer Phe Phe Asp Asn Thr Thr Thr Thr Val Glu Glu His Val 85 90 95 Pro PheArg Val Thr Trp Thr Asn Gly Thr Ile Pro Pro Pro Val Thr 100 105 110 GluAla Ser Ser Val Pro Lys Asn Asn Cys Leu Gln Gly Ile Glu Phe 115 120 125Leu Glu Glu Glu Asn Val Arg Ile Gly Ile Leu Phe Ala Ser Lys Ala 130 135140 Leu Met Gln Leu Leu Val Asn Pro Phe Val Gly Pro Leu Thr Asn Arg 145150 155 160 Ile Gly Tyr His Ile Pro Met Phe Val Gly Phe Met Ile Met PheLeu 165 170 175 Ser Thr Leu Met Phe Ala Phe Ser Gly Thr Tyr Ala Leu LeuPhe Val 180 185 190 Ala Arg Thr Leu Gln Gly Ile Gly Ser Ser Phe Ser SerVal Ala Gly 195 200 205 Leu Gly Met Leu Ala Ser Val Tyr Thr Asp Asn TyrGlu Arg Gly Arg 210 215 220 Ala Met Gly Ile Ala Leu Gly Gly Leu Ala LeuGly Leu Leu Val Gly 225 230 235 240 Ala Pro Phe Gly Ser Val Met Tyr GluPhe Val Gly Lys Ser Ser Pro 245 250 255 Phe Leu Ile Leu Ala Phe Leu AlaLeu Leu Asp Gly Ala Leu Gln Leu 260 265 270 Cys Ile Leu Trp Pro Ser LysVal Ser Pro Glu Ser Ala Met Gly Thr 275 280 285 Ser Leu Leu Thr Leu LeuLys Asp Pro Tyr Ile Leu Val Ala Ala Gly 290 295 300 Ser Ile Cys Leu AlaAsn Met Gly Val Ala Ile Leu Glu Pro Thr Leu 305 310 315 320 Pro Ile TrpMet Met Gln Thr Met Cys Ser Pro Glu Trp Gln Leu Gly 325 330 335 Leu AlaPhe Leu Pro Ala Ser Val Ala Tyr Leu Ile Gly Thr Asn Leu 340 345 350 PheGly Val Leu Ala Asn Lys Met Gly Arg Trp Leu Cys Ser Leu Val 355 360 365Gly Met Val Ala Val Gly Ile Ser Leu Leu Cys Val Pro Leu Ala His 370 375380 Asn Ile Phe Gly Leu Ile Gly Pro Asn Ala Gly Leu Gly Phe Ala Ile 385390 395 400 Gly Met Val Asp Ser Ser Leu Met Pro Ile Met Gly Tyr Leu ValAsp 405 410 415 Leu Arg His Thr Ser Val Tyr Gly Ser Val Tyr Ala Ile AlaAsp Val 420 425 430 Ala Phe Cys Val Gly Phe Ala Ile Gly Pro Ser Thr GlyGly Val Ile 435 440 445 Val Gln Val Ile Gly Phe Pro Trp Leu Met Val IleIle Gly Thr Ile 450 455 460 Asn 465

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of: (a) a polynucleotide encoding a polypeptide havingthe deduced amino acid sequence of SEQ ID No. 2 and fragments, analogsor derivatives of said polypeptide; and (b) a polynucleotide encoding apolypeptide having the amino acid sequence encoded by the cDNA containedin ATCC Deposit No. 75980 and fragments, analogs or derivatives of saidpolypeptide.
 2. The polynucleotide of claim 1 wherein the polynucleotideis DNA.
 3. The polynucleotide of claim 2 wherein said polynucleotideencodes a polypeptide having the deduced amino acid sequence of SEQ IDNo.
 2. 4. The polynucleotide of claim 2 comprising the nucleotidesequence from nucleotide 1 to nucleotide 2885 of SEQ ID No.
 1. 5. Avector containing the DNA of claim
 2. 6. A host cell geneticallyengineered with the vector of claim
 5. 7. A process for producing apolypeptide comprising: expressing from the host cell of claim 6 thepolypeptide encoded by said DNA.
 8. A process for producing cellscapable of expressing a polypeptide comprising genetically engineeringcells with the vector of claim
 5. 9. A polypeptide selected from thegroup consisting of (i) a polypeptide having the deduced amino acidsequence of SEQ ID No. 2 and fragments, analogs or derivatives thereofand (ii) a polypeptide encoded by the cDNA of ATCC Deposit No. 75980 andfragments, analog or derivatives of said polypeptide.
 10. An antibodyagainst the polypeptide of claim
 9. 11. A compound effective as anagonist to the polypeptide of claim
 9. 12. A compound effective as anantagonist against the polypeptide of claim
 9. 13. A method for thetreatment of a patient having need of human amine transporter activitycomprising: administering to the patient a therapeutically effectiveamount of the compound of claim
 11. 14. A method for the treatment of apatient having need of human amine transporter activity comprising:administering to the patient a therapeutically effective amount of thepolypeptide of claim 9, wherein said therapeutically effective amount ofthe polypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 15. A methodfor the treatment of a patient having need to inhibit human aminetransporter activity comprising: administering to the patient atherapeutically effective amount of the compound of claim
 12. 16. Asoluble fragment of the polypeptide of claim 9 wherein the polypeptidebinds a ligand for the receptor.
 17. A process for identifying compoundseffective as antagonists or agonists to the a polypeptide of claim 9comprising: expressing the polypeptide on the surface of a cell;contacting the cell with a ligand known to be transported by thepolypeptide and a compound to be screened; determining the extent ofligand transported by the polypeptide; and identifying if the compoundto be screened is an agonist or antagonist.
 18. A process fordetermining whether a ligand not known to be capable of binding to thepolypeptide of claim 9 can bind thereto comprising: contacting amammalian cell which expresses the human amine transporter with apotential ligand; detecting the presence of the ligand which binds tothe transporter; and determining whether the ligand binds to thetransporter.
 19. A method for diagnosing a disease or a susceptibilityto a disease related to under-expression of the polypeptide of claim 9comprising: detecting mutations in the nucleic acid sequence encodingthe polypeptide of claim 14 in a sample derived from a host.
 20. Adiagnostic process comprising: analyzing for the presence of thepolypeptide of claim 22 in a sample derived from a host.